Composition for the preservation of viruses

ABSTRACT

This invention relates to a composition for the preservation of a virus, the composition including a virus, a lipid and a cryoprotectant.

This nonprovisional application claims priority under 35 U.S.C. § 119(a)on Patent Application No. PR9449 filed in Australia on Dec. 12, 2001 andPS0545 filed in Australia on Feb. 14, 2002, which is herein incorporatedby reference.

FIELD OF THE INVENTION

The present invention relates to compositions for the preservation ofviruses. The present invention also relates to methods for preparingcompositions for the preservation of viruses.

It will become apparent from the following description that the viralcompositions according to the present invention are most likely to bepharmaceutical compositions for the purposes of the delivery of viralparticles for gene therapy or vaccination. However, it must beappreciated that the invention is not to be limited in its applicationto only pharmaceutical compositions.

BACKGROUND OF THE INVENTION

Gene therapy broadly refers to the transfer of genetic material intocells and the expression of that material in those cells for atherapeutic purpose. The goal is to produce the desired protein in theappropriate quantity and the proper location. Although a variety ofmethods have been developed to deliver therapeutic nucleic acids tocells, many of these methods are limited by relatively inefficienttransfer of the therapeutic nucleic acid to the target cells. Becauseviruses are highly efficient at infecting susceptible cells, viruses arenow recognised as being useful vehicles for the transfer of therapeuticnucleic acids into cells for the purpose of gene therapy.

Viruses fall broadly into two distinct groups: those that integrate intothe genome of transduced cells and those that do not. An integratingvirus inserts its viral genome into host DNA to facilitate long-termgene expression. For a non-integrating virus, however, the viral genomeexists extra-chromosomally as an episome in the nucleus of transducedcells. Depending on the ability of the virus to replicate, the viralgenome is either passed on faithfully to every daughter cell or iseventually lost during cell division.

Retroviruses and adeno-associated viruses (AAVs) may integrate into thehost DNA to provide a steady level of expression following transductionand incorporation into the host genome. As the target DNA is replicated,so too is the inserted therapeutic gene embedded in the transferredchromosomal DNA. Thus, transduction via these vectors can producedurable gene expression. This can be advantageous in tumour vaccinestrategies in which a steady level of gene expression may enhanceefficacy.

In contrast, adenovirus and vaccinia virus vectors do not integrate intothe host DNA but exist as episomes. Thus, a transferred gene isexpressed without actual integration of the gene into the target cellgenome. Generally, non-integrating viruses are used when transient geneexpression is desired.

Examples of viruses that may be used to deliver nucleic acids to cellsfor gene therapy purposes include adenovirus, adeno-associated virus(AAV), retrovirus, herpes simplex virus, vaccinia virus, poliovirus,sindbis virus, HIV-1, avian leukosis virus, sarcoma virus, Epstein-Barrvirus, papillomavirus, foamy virus, influenza virus, Newcastle diseasevirus, sendai virus, lymphocytic choriomeningitis virus, polyoma virus,reticuloendotheliosis virus, Theiler's virus, and other types of RNA andDNA viruses.

The use of attenuated and killed viruses for purposes of vaccination isalso well known. In addition, viruses are also becoming increasinglyimportant as tools for research and diagnostics. The increasingimportance of viruses as tools for gene therapy, vaccination, andresearch and diagnosis has led to a need to develop viral compositionsthat may be manufactured, stored and used without compromising viralefficacy. For example, viral compositions for vaccination must be ableto maintain the immunogenicity of a virus, or the immunogenicity of acomponent of the virus. In the case of compositions of viruses to beused for gene therapy, it is critical that the efficacy of the liveviral formulations carrying therapeutic transgenes be maintained.

Because viruses are biological entities consisting of a nucleic acidencapsulated by a protein coat, they are susceptible to the samechemical and physical processes that may degrade or inactivate proteinsand nucleic acids. In particular, live viruses may often be verysusceptible to damage, as any change in the conformation or integrity ofone or more components of the virus coat or the encapsulated nucleicacid may lead to a loss of infectivity. As such, biopharmaceuticalproducts containing compositions of viruses for vaccination or genetherapy usually require stringent conditions to avoid physicochemicaldegradation and to maintain biological activity. Degradation of virusesin such compositions may occur during isolation, production,purification, formulation, storage, shipping or delivery of the virus.Accordingly, biopharmaceutical compositions of viruses must beformulated to provide protection of the virus against factors such astemperature, pH, pressure, oxidising agents, ionic content, light,radiation, ultrasound, and changes in phase (for example as occursduring freezing and thawing (“freeze-thawing”).

In addition to the factors already discussed, other factors such asviral concentration, the size and structure of the encapsulated nucleicacid, container composition, headspace gas, and number of freeze-thawcycles may all affect the activity of viral compositions.

As a consequence, the utility of many viruses in biopharmaceuticalpreparations is often limited by the instability of compositions of theviruses, particularly upon storage. For example, even when viralcompositions are stored at very low temperature (for example −80° C.) inthe frozen state, a significant loss of infectivity may still occur overtime. A further loss of infectivity may occur upon thawing of the frozenviral composition.

In addition, as low temperature storage conditions are not alwaysavailable, it would be advantageous to develop formulations that improvethe preservation of frozen viral formulations above −80° C. for extendedperiods of time, such as extended storage at temperatures just belowfreezing. Indeed, viral compositions that must be stored at very lowtemperature and cannot be stored at standard freezer temperatures (forexample −10° C. to −20° C.) for substantial periods of time represent aserious impediment to the widespread clinical use of many viruses.

As will be also appreciated, the storage of products at standard freezertemperatures may also be problematic, because often such freezersundergo temperature cycling that may result in the viral compositionbeing subjected to temperatures above freezing, and as such thecompositions may undergo repeated cycles of freezing and thawing.Freeze-thawing may also occur during large scale production, handling ordistribution.

It would also be advantageous to develop viral compositions that canmaintain the desired pH of the composition for extended periods of timedespite being exposed to refrigeration temperatures and/or subjected toconditions such as freeze-thawing, especially the slow rate offreeze-thawing that may occur during large scale production, handling ordistribution.

Finally, increasingly high concentrations of virus are also beingrequired for therapeutic purposes. However, the concentration of virusin a composition may present additional problems to the ability topreserve a virus. In particular, a high concentration of virus maycontribute significantly to viral instability due to aggregation and/orprecipitation.

Therefore for many viruses a deficiency has been the inability toformulate compositions that acceptably preserve the virus, particularlyin the frozen state. Such deficiencies with the ability to preserve theactivity of viral compositions often preclude their use for genetherapy, for vaccination, or for other purposes.

It is therefore an aim of the present invention to provide a compositionfor the improved preservation of viruses.

Throughout this specification reference may be made to documents for thepurpose of describing various aspects of the invention. However, noadmission is made that any reference cited in this specificationconstitutes prior art. In particular, it will be understood that thereference to any document herein does not constitute an admission thatany of these documents forms part of the common general knowledge in theart in Australia or in any other country. The discussion of thereferences states what their authors assert, and the applicant reservesthe right to challenge the accuracy and pertinency of any of thedocuments cited herein.

SUMMARY OF THE INVENTION

The present invention provides a composition for the preservation of avirus, the composition including a virus, a lipid and a cryoprotectant.

The present invention further provides a method of producing acomposition for the preservation of a virus, the method including thestep of preparing a liquid composition including a virus, a lipid and acryoprotectant.

In the context of the present invention, it has been determined that theactivity of a virus in a composition may be preserved by including inthe composition a lipid and a cryoprotectant.

The composition according to the present invention provides for improvedpreservation of a virus. For example, the composition provides forimproved storage of a virus upon storage. The composition also providesfor improved preservation of a virus upon freezing and provides forimproved preservation of the virus upon storage of the composition inthe frozen state. The composition further provides for improvedpreservation of a virus upon thawing. The improved preservation of thevirus in the composition is also evident over a broad range of storagetemperatures and storage periods, and over multiple cycles of freezingand thawing (“freeze-thawing”).

It is to be understood that while the composition according to thepresent invention may be used for the preservation of viable virusparticles, the composition according to the present invention may alsobe used for the improved preservation of attenuated virus particles,killed virus particles, non-viable viral particles, synthetic viruses,or one or more constituents of viable, killed, non-viable or syntheticviruses.

It will also be appreciated that not only may the present invention beused for pharmaceutical compositions for medical applications, such asthe delivery of virus for the purposes of gene therapy or the deliveryof viruses or viral constituents for vaccination, the present inventionmay also be used for compositions for the preservation of viable,attenuated, killed, non-viable and synthetic viral particles fornon-medical applications, such as the preservation of viral preparationsfor research and diagnostic applications.

Various terms that will be used throughout this specification havemeanings that will be well understood by a skilled addressee. However,for ease of reference, some of these terms will now be defined.

The term “preservation” as used throughout the specification is to beunderstood to mean that a desired activity of a virus (such asinfectivity, transduction or immunogenicity) does not decreasesubstantially over a given period of time, or that a desired activity ofa virus does not decrease substantially after a particular treatment.For example (i) the activity of a virus in a composition according tothe present invention may not decrease substantially when the virus isstored in a frozen composition for a given period of time; and/or (ii)the activity of the virus in a composition according to the presentinvention may not substantially decrease when the composition isfreeze-thawed, or subjected to repeated cycles of freeze-thawing.

In the context of the present invention, the ability of a composition topreserve a virus is to be understood to be improved over similarcompositions that do not contain a lipid, or compositions that do notcontain a cryoprotectant. Accordingly, the composition according to thepresent invention will show an activity of the virus over a given periodof time, or will show an activity of the virus after a particulartreatment (eg freeze-thawing), that is higher than a similar compositionthat does not contain a lipid, or a similar composition that does notcontain cryoprotectant.

In this regard, the demonstration of the preservation of a virus in acomposition according to the present invention will be achieved by asuitable biological assay. As will be appreciated, given the degree ofvariability in biological systems, in determining the ability of acomposition to preserve a virus, sufficient repetitions of anybiological assay will need to be performed to statistically demonstratethat the composition is able to preserve the virus.

The term “virus” as used throughout the specification is to beunderstood to mean any natural, recombinant, in vitro packaged orsynthetic virus.

The term “viral composition” as used throughout the specification is tobe understood to mean any composition that may be used for thepreservation of a virus (or a part of a virus) for therapeutic purposes,or for the preservation of virus (or part of a virus) generally. Theterm not only encompasses the composition according to the presentinvention, but also encompasses the composition according to the presentinvention with other any other additives, such as excipients.

The term “lipid” as used throughout the specification is to beunderstood to mean any fatty acid and derivatives of fatty acids,glycerol-derived lipids including phospholipids, sphingosine-derivedlipid (including ceramides, cerebrosides, gangliosides andsphingomyelins) and glycolipids, terpenes and their derivatives, longchain alcohols and waxes. In referring to such lipids, it will beappreciated that these molecules are amphiphilic and will contain asubstantially hydrophilic moiety coupled to a substantially hydrophobicmoiety. The hydrophilic moiety will contain one or more substantiallyhydrophilic groups, and the hydrophobic moiety will contain one or moresubstantially hydrophobic groups.

The term “cryoprotectant” as used throughout the specification is to beunderstood to mean any molecule that has the function of substantiallyinhibiting the formation of ice crystals upon freezing of a liquidcomposition. In this regard, it will be understood that a molecule withcryoprotective function may also perform one or more additionalfunctions in any particular composition (for example being a tonicitymodifier or lyoprotectant). Accordingly, the demonstration that amolecule has a cryoprotectant capacity will be achieved by a suitablemethod known in the art to test whether the molecule has the ability toinhibit the formation of crystals upon the freezing of a liquidcomposition.

The term “surfactant” as used throughout the specification is to beunderstood to mean any compound that can reduce the interfacial tensionbetween two immiscible phases. In this regard, it will be understoodthat a molecule with surfactant function may also perform one or moreadditional functions in any particular composition. Accordingly, thedemonstration that a molecule has a surfactant capacity will be achievedby a suitable method known in the art to test whether the molecule hasthe ability to reduce the interfacial tension between two immisciblephases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: shows the storage stability of various viral compositions at pH8and −80° C.

FIG. 2: shows the storage stability of various viral compositions at pH6and −80° C.

FIG. 3: shows the storage stability of various viral compositions at pH8and −20° C.

FIG. 4: shows the storage stability of various viral compositions at pH6and −20° C.

FIG. 5: shows the effect of multiple freeze-thaw cycles on various viralcompositions.

FIG. 6: shows the effects of multiple freeze-thaw cycles on OAdV623 intris/sucrose/PEG400.

FIG. 7: shows the effects of multiple freeze-thaw cycles on OAdV623 intris/sucrose/lipid/PEG400.

GENERAL DESCRIPTION OF THE INVENTION

As mentioned above, the composition of the present invention providesfor the improved preservation of virus particles. Preferably, the virusparticles are selected from one or more of the group consisting ofAdenoviridae including Mastadenovirus such as Human Adenovirus andAtadenovirus such as Ovine Adenovirus; Herpesviridae; Poxviridaeincluding vaccinia, fowlpox, swinepox and sheeppox; Papovaviridae;Orthohepadnavirus; Parvoviridae including adeno-associated virus;Birnaviridae; Reoviridae; Flaviviridae; Picornaviridae includingpoliovirus; Togaviridae including Sindbis virus and Semliki Forestvirus; Filoviridae; Paramyxoviridae; Rhabdoviridae; Arenaviridae;Bunyaviridae; Orthomyxoviridae; Retroviridae including Lentivirus. Morepreferably, the virus particle is derived from the Adenoviridae familyof viruses.

More preferably, the virus is an Atadenovirus. Most preferably, thevirus is an ovine atadenovirus.

For the purposes of the various forms of the present invention, thevirus is preferably a recombinant virus. More preferably, the virus is arecombinant virus that has utility for the purposes of gene therapy. Ina particularly preferred embodiment, the virus is a recombinant ovineadenovirus, such as the adenoviral vector OAdV623 or derivatives of thisvector. OAdV623 encodes the purine nucleoside phosphorylase (PNP) genewhich catalyses the conversion of the immunosuppressive prodrugFludarabine to the toxic 2-fluoro-adenine product. Adenoviral vectorOAdV623 is as described in Lockett L. J. and Both G. W. (2002) Virology294:333–341.

The composition according to the present invention may be used for thepreservation of viral particles that retain the ability to infect ortransduce cells, or for the preservation of viral particles that havebeen attenuated, killed, are non-viable, have been produced by in vitropackaging or are of synthetic origin. The composition according to thepresent invention may also be used for the preservation of parts of avirus, such as the preservation of one or more constituents of the viruscoat. Preferably, the viral particles are viable viral particles.

In this regard, an attenuated virus is to be understood to mean a viruswhose virulence has been lowered by a biological, physical or chemicalprocess. For example, the virulence of a virus may be attenuated bypassaging through a semi-permissive host.

A killed virus is to be understood to mean a viral particle that hasbeen inactivated by a treatment so that the viral particle no longerretains the ability to infect a permissive host. Examples of treatmentsthat may kill a viral particle are heat or chemical modification.

A non-viable virus is to be understood to mean a viral particle that isnot able to infect or transduce permissive host cells.

A synthetic virus is to be understood to mean any nucleic acid packagedwith a protein and/or lipid coat.

The composition according to the present invention may be used for thepreservation of viruses that are to be used for medical applications.Preferably, the composition is for the preservation of viruses that areto be used for the purposes of gene therapy. More preferably, thecomposition is for the preservation of viruses that are to be used forthe delivery of therapeutic nucleic acids to prostatic cells for genetherapy.

The composition according to the present invention may also be used forthe preservation of viruses that are to be used for the purposes ofeliciting an immunogenic response, such as for vaccination. It will beunderstood in this regard that the composition may be used for thepreservation of whole viruses, or for the preservation of one or moreimmunogenic constituents of a virus, such as the preservation of one ormore polypeptides that make up part of the virus coat.

When the composition according to the present invention is used for thepreservation of a virus to be used for medical applications, thecomposition may also include one or more pharmaceutically acceptableadditives, such as pharmaceutically acceptable salts, amino acids,polypeptides, polymers, solvents, buffers and bulking agents.

The composition according to the present invention is preferably aliquid composition. The liquid composition may be a substantiallyaqueous composition or a composition composed of one or more othersolvents. Most preferably, the composition is a substantially aqueouscomposition.

The composition may be stored in a container suitable for thepreservation of the virus, such as borosilicate glass. The compositionmay also be stored under a gaseous atmosphere that is suitable for thepreservation of the virus including air, argon or nitrogen.

The composition according to the present invention may also be used forthe preservation of viable, attenuated, killed, non-viable or syntheticviruses for research applications. For example, the composition may beused for the preservation of viral particles that have use in researchapplications, such as the use of viral preparations for immunologicalresearch. The composition may also be used for the preservation of viralpreparations for use in molecular biological research, such as the useof viral preparations for the infection or transduction of cells inculture.

In a similar fashion, the composition according to the present inventionmay also be used for the preservation of viral particles that have usein diagnostic applications, such as the use of viral preparations aspositive and negative test standards for diagnostic applications.

With regard to viral activity, the activity of the virus may be measuredby any suitable assay that is known in the art. Such assays include bothdirect and indirect biological and physicochemical assays of viralactivity. Examples of direct assays include the measurement of thenumber of infectious viral particles in the product, the expression of areporter gene or other transgene carried by the virus, the cell killingor cell viability following viral infection or transduction of asuitable cell line, or the quantity of components produced followingadministration of the viral particles or constituents to a suitablemodel (eg. immune response in case of vaccination). Examples of indirectassays include the measurement of the number of intact andnon-aggregated viral particles or the size of the viral particles (as anindication of viral aggregation) in the product.

For example, for determining the activity of viable viral particles, thenumber of permissive cells killed following infection or transductionwith a defined amount of virus may be determined by any suitable assay.Alternatively, as an indirect measure of viral activity, the number ofintact and non-aggregated viral particles in the product may bedetermined by anion-exchange HPLC and the particle size determined bylight scattering analysis.

The concentration of virus in the composition of the present inventionmay also affect the ability of the composition to preserve the virus.Preferably, the concentration of virus in the composition is in therange from 1×10⁶ to 1×10¹⁴ virus particles/ml. More preferably, theconcentration of virus is in the range from 1×10⁸ to 5×10¹² virusparticles/ml.

The lipid in the composition according to the present invention is anyfatty acid or derivative of a fatty acid, glycerol-derived lipidincluding a phospholipid, sphingosine-derived lipid (includingceramides, cerebrosides, gangliosides and sphingomyelins) andglycolipid, terpene and their derivatives, long chain alcohol and wax.The lipid is an amphiphilic molecule that contains a substantiallyhydrophilic moiety coupled (directly or by way of a spacer) to asubstantially hydrophobic moiety. The hydrophilic moiety will containone or more substantially hydrophilic groups and the hydrophobic moietywill contain one or more substantially hydrophobic groups.

The lipid present in the composition according to the various forms ofthe present invention may be a cationic lipid, anionic lipid,zwitterionic lipid, non-ionic lipid or any combination of such lipids.

Examples of cationic lipids include2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA), dioctadecylaminoglycyl spermine (DOGS),dipalmitoyl phosphatidylethanolamyl spermine (DPPES),1,3-dioleoyoxy-2-(6-carboxy-spermyl)-propylamide (DOSPER),dioleyldimethylammonium chloride (DODAC),N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimetylammonium chloride (DOTMA),1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP),1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide(DMRIE), 3β-(N-((N′, N′-dimethylamino)ethane)carbamoyl)-cholesterol(DC-Chol), dimethyldioctadecyl ammonium bromide (DDAB),1-[2-(oleoyoxy)-ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride(DOTIM), bis(oleoyl)-trimethylaminomethylphosphonate,1,2-dimyristoylglycerolpentalysine salt,N,N′,N″,N′″-tetramethyl-N,N′,N″,N′″-tetrapalmitylspermine (TMTPS),cetyltrimethylammonium bromide (CTAB) and the following proprietarycationic lipids: Lipofectamine (DOSPA:DOPE 3:1 w/w), Lipofectin(DOTMA:DOPE 1:1 w/w), Lipofectace (DDAB:DOPE 1:1.25 w/w), Transfectam,Cellfectin (TMTPS:DOPE 1:1.5 M/M), Superfect, LipoTaxi, DMRIE-C(DMRIE/cholesterol: 1:1) and trilysine-carpryloyl-tris-trilaurate(T-shape; CS087).

Examples of anionic lipids include1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS),1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), and PEG-PE lipidssuch as1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethyleneglycol)2000] (PEG2000 DMPE),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethyleneglycol)2000] (PEG2000 DPPE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethyleneglycol)2000] (PEG2000 DSPE).

Examples of zwitterionic/neutral lipids include1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DM PE).

Preferably the lipid is a cationic lipid. More preferably, the lipid isa cationic lipid that has a hydrophilic moiety that includes one or moreamino residues. More preferably, the lipid is a cationic lipid that hasa hydrophilic moiety that includes one or more groups derived from aminoacids. More preferably, the lipid is a cationic lipid that has ahydrophilic moiety that includes one or more groups derived from apositively charged amino acid, such as lysine, arginine or histidine.Most preferably, the lipid is a cationic lipid that has a hydrophilicmoiety including one or more lysine groups.

In a particularly preferred embodiment, the lipid is a poly-cationiclipid. Preferably, the lipid is a poly-cationic lipid that has ahydrophilic moiety that includes two or more amino residues. Morepreferably, the lipid is a poly-cationic lipid that has a hydrophilicmoiety that includes two or more groups derived from amino acids. Morepreferably, the lipid is a poly-cationic lipid that has a hydrophilicmoiety that includes two or more groups derived from positively chargedamino acids, such as lysine, arginine or histidine. Most preferably, thelipid is a poly-cationic lipid that has a hydrophilic moiety thatincludes three lysine groups.

The hydrophobic moiety of the lipid in the composition according to thepresent invention includes one or more hydrophobic groups. Hydrophobicgroups include, but are not restricted to, acyl, alkyl, or alkoxychains. Preferably, the one or more hydrophobic groups are derived froman acyl group of a fatty acid. More preferably, the one or more acylgroups have a carbon chain length of 3 to 24 carbon atoms. Mostpreferably, the one or more acyl groups is a laurate group.

Preferably, the lipid in the composition according to the presentinvention has a hydrophobic moiety that includes two or more hydrophobicgroups. More preferably the lipid has a hydrophobic moiety that includesthree hydrophobic groups. Most preferably, the lipid has a hydrophobicmoiety that includes three laurate groups.

The lipid in the composition according to the present invention may alsoinclude a spacer group between the hydrophilic moiety and thehydrophobic moiety. The spacer group may include any combination orseries of atoms that covalently join the hydrophilic and hydrophobicmoieties. Preferably, the spacer region has a chain length equivalent to1 to 30 carbon-carbon single covalent bonds.

In a preferred embodiment, the lipid in the composition according to thevarious forms of the present invention is derived from a tris-conjugatedcationic lipid (or a salt thereof) according to the following generalformula:

In this general formula, X represents the hydrophilic moiety, Yrepresents a spacer group (which may or may not be present), and R₁, R₂and R₃ are acyl groups of fatty acids. Preferably, a spacer group Y ispresent in the molecule. Most preferably the spacer group has a chainlength equivalent to 1 to 30 carbon-carbon single covalent bonds.

Most preferably, the lipid in the composition according to the variousforms of the present invention is the moleculetrilysine-carpryloyl-tris-trilaurate (T-shape; CS087), or a saltthereof, the structure of which is as follows:

To preserve virus, the concentration of the lipid in the composition ispreferably in the range from 0.1 μM to 1 mM. More preferably theconcentration of the lipid is 1 μM to 500 μM. In the most preferredembodiment, the concentration of the lipid is 5 μM to 100 μM.

In the case where the lipid in the composition istrilysine-carpryloyl-tris-trilaurate (CS087), the concentration of thelipid is preferably in the range from 10 to 50 μM. Most preferably, theconcentration of trilysine-carpryloyl-tris-trilaurate in the compositionis 10 μM.

The cryoprotectant in the composition according to the various forms ofthe present invention is any molecule that has the function ofsubstantially inhibiting the formation of ice crystals upon freezing ofa liquid. The cryoprotectant may be a sugar, sugar alcohol, glycerol,amino acid including glycine, histidine or arginine, peptide,polypeptide, protein including albumin and gelatine, or polymerincluding dextran, polyvinyl pyrrolidone, polyvinyl alcohol orpolyethylene glycol, or any combination of such molecules.

The determination of whether a molecule may function as a cryoprotectantmay be by a suitable method known in the art in which the function of amolecule to substantially inhibiting the formation of ice crystals uponfreezing of a liquid may be tested.

In a preferred embodiment of the invention, the cryprotectant is apoly-hydroxy compound. More preferably, the poly-hydroxy compound is asugar. More preferably, the sugar is sucrose, trehalose, dextrose,lactose, maltose or glucose. In a particularly preferred embodiment, thecryoprotectant is sucrose.

To preserve virus, the concentration of the cryoprotectant in thecomposition may be in the range of 0.1 to 20% weight/volume. Preferably,the concentration is in the range of 1 to 10% weight/volume. When thecryoprotectant present in the composition is sucrose, preferably theconcentration of sucrose is 8.5%.

It has also been found that the presence of a surfactant in thecomposition may further improve the ability of the composition topreserve a virus. The surfactant is any molecule that can reduce theinterfacial tension between two immiscible phases. Preferably thesurfactant is a non-ionic surfactant.

The determination of whether a molecule may function as a surfactant maybe by a suitable method known in the art in which the function of amolecule to reduce the interfacial tension between two immiscible phasesmay be tested.

Preferably, the surfactant is present in the composition at aconcentration in the range from 0.0001% to 50% volume/volume. Morepreferably, the surfactant is present in the composition at aconcentration in the range from 0.001% to 10% volume/volume.

In a preferred embodiment, the non-ionic surfactant is a molecule thatincludes an oxyethylene group and a hydroxy group. Most preferably, thenon-ionic surfactant is polysorbate 80 or polyethylene glycol 400, orany combination of these non-ionic surfactants.

When polysorbate 80 is used in the composition, the concentration of thepolysorbate 80 is preferably 0.0001 to 1% volume/volume. Morepreferably, the concentration of polysorbate 80 in the composition is0.001 to 0.1% volume/volume. Most preferably, the concentration ofpolysorbate 80 in the composition is 0.005% volume/volume.

When polyethylene glycol 400 is used in the composition, theconcentration is preferably 0.001 to 50% volume/volume. More preferably,the concentration of polyethylene glycol 400 is 0.01 to 10%volume/volume. More preferably, the concentration of polyethylene glycol400 is 0.01 to 5% volume/volume. Most preferably, the concentration ofpolyethylene glycol 400 in the composition is 0.5% volume/volume.

The pH of the composition may also be selected to improve viralpreservation. The pH may also be selected to be compatible with theadministration of the composition to a subject for therapeutic purposes.Preferably, the pH of the composition is in the range of 4 to 10. Morepreferably, the pH is in the range of to 9. In the most preferred formof the invention, the pH of the composition is in the range of 6 to 8.5.

The pH of the composition according to the various forms of the presentinvention may be obtained by buffering with a pharmaceuticallyacceptable buffer. Preferably, the buffer is selected from one or morebuffers selected from the group consisting of monobasic acids includingacetic, benzoic, gluconic, glyceric and lactic acids, dibasic acidsincluding aconitic, adipic, ascorbic, carbonic, glutamic, maleic, malic,succinic, tartaric acids, polybasic acids including citric andphosphoric acids. The buffer may also be selected from one or morebuffers selected from the group consisting of bases including ammonia orammonium chloride, diethanolamine, glycine, tromethamine (also known asTris and Tham).

Preferably, the buffer is selected from one or more buffers selectedfrom the group consisting of a tris-based buffer, a sodium hydrogenmaleate buffer, succinate buffer, or phosphate buffer. Tris-basedbuffers and sodium hydrogen maleate buffers are particularly preferred.

The composition according to the present invention provides acomposition for the improved preservation of a virus. In one embodiment,the present invention provides a composition for the preservation of avirus, the composition including a virus, a lipid and a cryoprotectant,wherein when the composition is frozen the virus is storage stable.

Preferably, the temperature of storage of the composition is from −200°C. to 0° C. More preferably, the temperature of storage is −100° C. to−5° C. Most preferably, the temperature of storage is −80° C. to −20° C.

With respect to the period of time over which the composition accordingto the present invention shows improved storage stability, thecomposition according to the present invention may be stored for aperiod of greater than 24 months. Preferably the period of storage is 12months or greater. More preferably, the period of storage is 6 months orgreater. More preferably, the period of storage is 3 months or greater.More preferably, the period of storage is 1 week or greater. Mostpreferably, the period of storage is 1 day or greater.

In this regard, as has been discussed previously, the improvedpreservation will be as compared to a composition that does not containlipid, or a composition that does not contain cryoprotectant. That is,the activity of the virus will not decrease substantially with time whenthe composition is stored at the abovementioned temperatures or for theabovementioned periods of time, as compared to a composition notcontaining lipid, or a composition not containing a cryoprotectant. Theactivity of the virus may be a desired activity of the virus in thecomposition, such as infectivity, ability to transduce orimmunogenicity.

The composition according to the present invention also shows improvedpreservation of a virus when the composition is frozen. The compositionalso shows improved preservation of a virus when the composition isthawed, or when the composition is subjected to one or more cycles offreezing and thawing. In a preferred embodiment, the present inventionprovides a composition for the preservation of a virus, the compositionincluding a virus, a lipid and a cryoprotectant, wherein the virus isstable to freeze-thawing.

The improved preservation with regard to freeze-thawing will be ascompared to a composition that does not contain lipid. That is, theactivity of the virus will not decrease substantially with time when thecomposition is frozen and thawed, or subjected to multiple cycles offreeze-thawing, as compared to a composition not containing lipid. Theactivity of the virus may be a desired activity of the virus in thecomposition, such as infectivity, ability to transduce orimmunogenicity.

The composition according to the present invention may also be in adosage form suitable for administration to a human or an animal subject.The dosage form includes the composition according to the presentinvention and may further include other pharmaceutically acceptableadditives.

The addition of such pharmaceutically acceptable additives to the dosageform may be to improve the ability of the virus to infect or transducetarget cells, or to improve the activity elicited by the administrationof virus. For example, local bystander killing can be enhanced byco-administration of a pharmaceutical or genetic agent which enhancescell-cell communications. Another example is the co-administration of aDNA encoding a cytokine to increase the immunogenicity of tumour cells.Another example is the inclusion of an adjuvant compound in a vaccine toenhance immune response.

The present invention also provides a method of producing a compositionfor the preservation of a virus, the method including the step ofpreparing a liquid composition including a virus, a lipid and acryoprotectant.

As will be appreciated, the methods according to the present inventionwill embody the same preferred features as those for the composition asdiscussed in detail above.

With regard to the preparation of virus, the virus may be purified byany suitable means. Preferably, the virus is purified by achromatographic method including ion-exchange chromatography or HPLC, orcentrifugation including CsCl centrifugation, after the virus has beenrecovered from infected permissive cells and/or the supernatant thereof.Preferably, the virus is purified by a chromatographic method. Whenpurified by CsCl centrifugation, the virus is prepared after recoveryfrom infected permissive cells by centrifugation through a CsCl stepgradient and centrifugation to equilibrium on a CsCl gradient. Whenvirus is purified in this manner, the CsCl is preferably removed bycolumn chromatography.

Preferably, the concentrated virus so formed is diluted in a solutionthat includes a suitable buffer and a cryoprotectant. More preferably,the solution further includes a non-ionic surfactant. In a preferredembodiment, the concentrated virus is diluted in a solution including aTris buffer, sucrose and polyethylene glycol 400 and/or polysorbate 80.In a particularly preferred embodiment, the concentrated virus isdiluted in a solution (at pH 8.0) including 10 mM Tris buffer, 8.5%sucrose and 2% polyethylene glycol 400. Preferably, the solution (whichmay exist as a suspension) containing virus is then filtered to removeunwanted micro-organisms. Most preferably, the solution is filteredthrough a 0.2 micron membrane filter.

For the preparation of a composition according to the present invention,the lipid is preferably first dispersed in a solution identical to thatused for the dilution of virus. Preferably, the solution (which mayexist as a suspension) containing lipid is filtered to remove unwantedmicro-organisms. Most preferably, the solution is filtered through a 0.2micron membrane filter.

To prepare a composition for the preservation of virus, the dilutedsolution of virus (which may exist as a suspension) may then be combinedwith a solution containing lipid (which may also exist as a suspension),the relative proportions of each selected so as to achieve the desiredfinal concentrations of virus and lipid. Accordingly, the methodaccording to the present invention provides a method for producing acomposition for the preservation of a virus, wherein the composition isformed by combining a solution including a virus and a cryoprotectantwith a solution including lipid.

The composition so formed may be stored in a suitable closed container.Preferably the composition is stored in borosilicate glass vials. Inaddition, the composition may be stored under a suitable gas or mixtureof gases.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made to examples that embody the above generalprinciples of the present invention. However, it is to be understoodthat the following description is not to limit the generality of theabove description.

EXAMPLE 1

Preparation of a Composition for the Preservation of Virus

CsCl purified OAdV623 virus was suspended in a pH 8.0 buffer containing10 mM Tris, 8.5% sucrose, 2% PEG buffer, in a polypropylene tube, attwo-times the final concentration. CS087 was supplied as a freeze-driedsolid that was first dissolved in ethanol and the ethanol then removedto produce a film. The film was dispersed in a pH 8.0 buffer containing10 mM Tris, 8.5% sucrose, 2% polyethylene glycol 400, in a polystyrenetube, at two-times the final concentration.

The suspensions of OAdV623 and CS087 were filtered separately through a0.2 μm membrane filter. An equal volume of OAdV623 and CS087 werecombined aseptically. The suspension was then gently agitatedcontinuously at approximately 40 rpm for 60 to 90 minutes at 18° C.–20°C., to ensure viral mixing. The final product was then asepticallydispensed into washed and autoclaved Type I borosilicate glass vials andstored at the appropriate temperature.

EXAMPLE 2

Storage Stability of Various Viral Compositions at −80° C.

The stability of various OAdV623 compositions (approximately 6×10⁸VP/ml) stored at −80° C. was assessed by determining the extent of cellkilling after storage for 14 days, 1 month or 3 months. Cell killing wasdetermined for compositions stored at pH 8 and pH 6.

OAdV623 encodes the PNP gene which catalyses the conversion of theimmunosuppressive prodrug Fludarabine to the toxic 2-fluoro-adenineproduct. This results in the death of cells producing PNP and to alimited extent, cells in the vicinity with a near neighbour bystandereffect. The death of susceptible cells, such as the PC3 cell line,following transduction with OAdV623 and treatment with Fludarabinephosphate, is a direct indicator of the potency of the OAdV623preparation.

To determine the extent of cell killing, an aliquot of virus in therelevant composition was thawed and approximately 6×10⁶ virus particleswere used to transduce 1×10⁴ PC3 cells in culture. The ability of thevirus to kill PC3 cells by converting the prodrug fludarabine, suppliedto the cells as fludarabine phosphate, to active 2-fluoroadenine, wasthen determined quantitatively. Cell killing was determined by an MTSassay (Promega) to measure the number of viable cells in treated wellscompared to a standard curve of cells not treated with the virus.

The concentration of the various components used was as follows:

10 mM Tris

10 mM sodium hydrogen maleate

8.5% sucrose

50 μM CS087

2% (v/v) polyethylene glycol 400 (PEG400)

0.005% (v/v) polysorbate 80 (PS80)

The whole composition was buffered to the desired pH with Tris ormaleate buffer.

(a) Stability at pH 8 is shown in FIG. 1.

% Cell Killing Composition 14 days 1 month 3 months Tris/sucrose  41.4 ±13.9 −13.3 ± 30.3 −14.2 ± 16.0 Tris/sucrose/CS087  34.6 ± 19.3  52.6 ±14.6  0.1 ± 11.0 Tris/sucrose/CS087/ 62.1 ± 1.3 68.9 ± 7.9 53.5 ± 5.0PEG400 Tris/sucrose/CS087/PS80 63.3 ± 0.7 74.2 ± 0.8 48.2 ± 4.9Tris/sucrose/PEG400 52.8 ± 1.5  3.0 ± 40.2  4.0 ± 4.4 Tris/sucrose/PS8040.3 ± 7.3 15.1 ± 9.3  5.3 ± 17.4

As can be seen, OAdV623 stored in tris/sucrose at −80° C. was stable for2 weeks only. The addition of lipid to the tris/sucrose compositionenhanced the preservation of the virus, when the virus was stored at−80° C. for periods of at least 1 month and then subsequently thawed.The addition of a non-ionic surfactant to the tris/sucrose/lipidcomposition further enhanced the preservation of virus, such that thevirus remained active even after a storage period of 3 months and asubsequent freeze-thaw cycle. Thus the preservation of the virus afterstorage and subsequent thawing was improved by the addition of lipidand, in particular, lipid plus a non-ionic surfactant.

(b) Stability at pH 6 is shown in FIG. 2.

% Cell Killing Composition 14 days 1 month 3 months Maleate/sucrose−15.4 ± 1.8  7.9 ± 16.9 −12.8 ± 19.8 Maleate/sucrose/CS087  −0.2 ± 4.166.8 ± 5.7  18.9 ± 15.2 Maleate/sucrose/CS087/  9.1 ± 3.5 71.7 ± 6.228.5 ± 4.6 PEG400 Maleate/sucrose/CS087/  23.4 ± 1.1 75.2 ± 6.5 48.6 ±3.5 PS80 Maleate/sucrose/PEG400 −15.2 ± 1.8 20.9 ± 8.7  2.7 ± 6.3Maleate/sucrose/PS80 −15.4 ± 1.0 24.1 ± 6.5 −4.0 ± 7.9

As can be seen, the addition of lipid to the tris/sucrose compositionenhanced the preservation of the virus, when the virus was stored at−80° C. such that the virus remained active after a storage period of 3months and a subsequent freeze-thaw cycle. The addition of a non-ionicsurfactant to the tris/sucrose/lipid composition further enhanced thepreservation of virus. Thus the preservation of the virus after storageand subsequent thawing was improved by the addition of lipid and, inparticular, lipid plus a non-ionic surfactant.

EXAMPLE 3

Storage Stability of Various Viral Compositions at −20° C.

The stability of various OAdV623 compositions (approximately 6×10⁸VP/ml) stored at −20° C. was assessed by determining the extent of cellkilling after storage for 14 days, 1 month or 3 months. Cell killing wasdetermined for formulations stored at pH 8 and pH 6.

To determine the extent of cell killing, an aliquot of virus in therelevant composition was thawed and approximately 6×10⁶ virus particleswere used to transduce 1×10⁴ PC3 cells in culture. The ability of thevirus to kill PC3 cells by converting the prodrug fludarabine, suppliedto the cells as fludarabine phosphate, to active 2-fluoroadenine, wasthen determined quantitatively. Cell killing was determined by an MTSassay (Promega) to measure the number of viable cells in treated wellscompared to a standard curve of cells not treated with the virus.

The concentration of the various components used was as follows:

10 mM Tris

10 mM sodium hydrogen maleate

8.5% sucrose

50 μM CS087

2% (v/v) polyethylene glycol 400 (PEG400)

0.005% (v/v) polysorbate 80 (PS80)

The whole composition was buffered to the desired pH with Tris ormaleate buffer.

(a) Stability at pH 8 is shown in FIG. 3.

% Cell Killing Composition 14 days 1 month 3 months Tris/sucrose 56.0 ±1.3  −7.6 ± 21.7 −14.2 ± 14.7 Tris/sucrose/CS087 57.3 ± 1.8  6.9 ± 12.9 13.5 ± 15.5 Tris/sucrose/CS087/ 67.2 ± 1.6 49.0 ± 7.5 66.8 ± 2.1 PEG400Tris/sucrose/CS087/PS80 65.5 ± 1.9 61.2 ± 5.0 57.2 ± 4.2Tris/sucrose/PEG400 60.5 ± 1.1 −11.2 ± 24.0  4.8 ± 11.7Tris/sucrose/PS80 55.9 ± 0.6  9.2 ± 15.8  9.4 ± 8.1

As can be seen, the addition of lipid to the tris/sucrose compositionenhanced the preservation of the virus, when the virus was stored at−20° C. such that the virus remained active after a storage period of 3months and a subsequent freeze-thaw cycle. The addition of a non-ionicsurfactant to the tris/sucrose/lipid composition further enhanced thepreservation of virus, such that greater activity (% cell kill) wasobserved compared to the tris/sucrose/lipid composition without anon-ionic surfactant. Thus the preservation of the virus after storageand subsequent thawing was improved by the addition of lipid and, inparticular, lipid plus a non-ionic surfactant.

(b) Stability at pH 6 is shown in FIG. 4.

% Cell Killing Composition 14 days 1 month 3 months Maleate/sucrose −7.1± 4.8  3.0 ± 10.8 −20.2 ± 14.2 Maleate/sucrose/CS087 16.4 ± 5.7 35.0 ±8.7  −2.6 ± 10.2 Maleate/sucrose/CS087/  29.4 ± 10.0  29.9 ± 18.0 −31.3± 10.8 PEG400 Maleate/sucrose/CS087/PS80 53.6 ± 1.5 66.1 ± 5.2 44.7 ±7.6 Maleate/sucrose/PEG400 12.4 ± 8.7  19.7 ± 17.7  1.9 ± 6.1Maleate/sucrose/PS80  3.1 ± 8.1 21.6 ± 9.7 −1.1 ± 4.6

The addition of lipid to the tris/sucrose composition enhanced thepreservation of the virus, when the virus was stored at −20° C. suchthat the virus remained active after a storage period of 1 month and asubsequent freeze-thaw cycle. The addition of a non-ionic surfactant tothe tris/sucrose/lipid composition further enhanced the preservation ofvirus. Thus the preservation of the virus after storage and subsequentthawing was improved by the addition of lipid and, in particular, lipidplus a non-ionic surfactant.

EXAMPLE 4

Preservation of Various Viral Compositions Upon Multiple Freeze-ThawCycles (−80° C. to 25° C.)

The ability of various OAdV623 compositions (1×10¹⁰ VP/ml) to bepreserved after multiple freeze-thaw cycles was assessed by determiningthe extent of cell killing after exposure to 1, 2 or 3 freeze-thawcycles. For each cycle, virus was frozen at −80° C. for no less than 1hour and thawed at 25° C. for 30 minutes. Cell killing was determinedfor virus formulated in 10 mM Tris, 8.5% sucrose, 50 μM CS087 and either2% or 4% polyethylene glycol 400 (pH 8).

To determine the extent of cell killing, virus particles in the range of4×10⁵ to 3×10⁷ were used to transduce 1×10⁴ PC3 cells in culture. Theability of the virus to kill PC3 cells by converting the prodrugfludarabine, supplied to the cells as fludarabine phosphate, to active2-fluoroadenine, was then determined quantitatively. Cell killing wasdetermined by an MTS assay (Promega) to measure the number of viablecells in treated wells compared to a standard curve of cells not treatedwith the virus.

The concentration of the various components used was as follows:

10 mM Tris

8.5% sucrose

50 μM CS087

2% or 4% (v/v) polyethylene glycol 400 (PEG400)

The whole composition was buffered to the desired pH with Tris ormaleate buffer.

The results are as shown in FIG. 5.

As can be seen, the composition containing either 2% or 4% polyethyleneglycol 400 provides substantial protection to the virus against theeffects of freeze-thawing of the composition. In particular, protectionagainst the effect of repeated freeze-thawing is most significant forthe composition containing 4% polyethylene glycol.

EXAMPLE 5

Preservation of Various Viral Compositions Upon Multiple Freeze-ThawCycles (−80° C. to 20° C.)

The ability of various OAdV623 compositions (approximately 9.6×10¹¹VP/ml) to be preserved after multiple freeze-thaw cycles was assessed bydetermining the extent of cell killing after exposure to 1 or 3freeze-thaw cycles. For each cycle, virus was frozen at −80° C. for atleast 1 hour and thawed at 20° C. for 40 minutes. Cell killing wasdetermined for virus formulated at pH 8 in 10 mM Tris, 8.5% sucrose and0.5% polyethylene glycol 400 with or without 10 μM CS087.

To determine the extent of cell killing, virus particles in the range of8×10⁵ to 1×10⁸ were used to transduce 5×10³ PC3 cells in culture. Theability of the virus to kill PC3 cells by converting the prodrugfludarabine, supplied to the cells as fludarabine phosphate, to active2-fluoroadenine, was then determined quantitatively. Cell killing wasdetermined by an MTS assay (Promega) to measure the number of viablecells in treated wells compared to control cells not treated with thevirus.

The concentration of the various components used was as follows:

10 mM Tris

8.5% sucrose

10 μM CS087

0.5% (v/v) polyethylene glycol 400 (PEG400)

The whole composition was buffered to the desired pH with Tris ormaleate buffer.

The results for OAdV623 in tris/sucrose/PEG400 is shown in FIG. 6 andOAdV623 in tris/sucrose/lipid/PEG400 is shown in FIG. 7.

As can be seen, the composition containing 10 μM lipid providessubstantial protection to the virus against the effects of repeatedfreeze-thawing compared to the composition containingtris/sucrose/PEG400 without any lipid.

Finally, it will be appreciated that various modifications andvariations of the described compositions and methods of the inventionwill be apparent to those skilled in the art without departing from thescope and spirit of the invention. Although the invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention, which are apparent tothose skilled in the field of virology, molecular biology, cryobiologyor related fields are intended to be within the scope of the presentinvention.

1. A composition for the preservation of a virus, said compositioncomprising a virus, a polycationic lipid that has a hydrophilic moietythat includes two or more groups derived from positively charged aminoacids, and a cryoprotectant, wherein the polycationic lipid has thefollowing chemical formula:

or a salt thereof, wherein X is a positively-charged hydrophilic moiety;Y is a spacer having a carbon chain length equivalent to 1 to 30carbon-carbon single covalent bonds or is absent; and R₁, R₂, and R₃ arethe same or different acyl groups of a fatty acid.
 2. A compositionaccording to claim 1, wherein the lipid has the following chemicalformula:


3. A composition according to claim 1, wherein the concentration oflipid is in the range from 0.1 μM to 1 mM.
 4. A composition according toclaim 1, wherein the concentration of lipid is in the range from 1 μM to500 μM.
 5. A composition according to claim 1, wherein the concentrationof lipid is in the range from 5 μM to 100 μM.
 6. A composition accordingto claim 2, wherein the concentration of lipid is 10 μM.
 7. Acomposition according to claim 1, wherein the cryoprotectant is apoly-hydroxy compound.
 8. A composition according to claim 7, whereinthe poly-hydroxy compound is a sugar.
 9. A composition according toclaim 8, wherein the sugar is sucrose, trehalose, dextrose, lactose,maltose, glucose or any combination of these sugars.
 10. A compositionaccording to claim 9, wherein the sugar is sucrose.
 11. A compositionaccording to claim 1, wherein the concentration of the cryoprotectant isin the range from 0.1 to 20% weight/volume.
 12. A composition accordingto claim 1, wherein the concentration of the cryoprotectant is in therange from 1 to 10% weight/volume.
 13. A composition according to claim10, wherein the concentration of sucrose is 8.5% weight/volume.
 14. Acomposition according to claim 1, wherein the composition furtherincludes a surfactant.
 15. A composition according to claim 14, whereinthe surfactant is a non-ionic surfactant.
 16. A composition according toclaim 15, wherein the non-ionic surfactant is a molecule that includesan oxyethylene group and a hydroxy group.
 17. A composition according toclaim 16, wherein the non-ionic surfactant is polysorbate 80 orpolyethylene glycol
 400. 18. A composition according to claim 14,wherein the concentration of the surfactant is in the range from 0.0001%to 10% volume/volume.
 19. A composition according to claim 17, whereinthe concentration of polysorbate 80 is in the range from 0.0001% to 1%volume/volume.
 20. A composition according to claim 17, wherein theconcentration of polysorbate 80 is 0.005% volume/volume.
 21. Acomposition according to claim 17, wherein the concentration ofpolyethylene glycol 400 is in the range from 0.01% to 10% volume/volume.22. A composition according to claim 17, wherein the concentration ofpolyethylene glycol 400 is 0.5% volume/volume.
 23. A compositionaccording to claim 1, wherein the virus is a virus derived from one ormore of the group consisting of Adenoviridae, Herpesviridae, Poxviridae,Papovaviridae, Orthohepadnavirus, Parvoviridae, Birnaviridae,Reoviridae, Flaviviridae, Picornaviridae, Togaviridae, Filoviridae,Paramyxoviridae, Rhabdoviridae, Arenaviridae, Bunyaviridae,Orthomyxoviridae, and Retroviridae.
 24. A composition according to claim23, wherein the virus is derived from the Adenoviridae family ofviruses.
 25. A composition according to claim 24, wherein the virus isan ovine atadenovirus.
 26. A composition according to claim 25, whereinthe virus is OAdV623 or a derivative of OAdV623.
 27. A compositionaccording to claim 1, wherein the concentration of virus in thecomposition is in the range from 1×10⁶ to 1×10¹⁴ virus particles/ml. 28.A composition according to claim 1, wherein the concentration of virusin the composition is in the range from 1×10⁸ to 5×10¹² virusparticles/ml.
 29. A composition according to claim 1, wherein the pH ofthe composition is in the range from 4 to
 10. 30. A compositionaccording to claim 1, wherein the pH of the composition is in the rangefrom 6 to 8.5.
 31. A composition according to claim 1, wherein the virusis storage stable.
 32. A composition according to claim 31, wherein thecomposition is stored at −200° C. to 0° C.
 33. A composition accordingto claim 31, wherein the composition is stored at −80° C. to −20° C. 34.A composition according to claim 31, wherein the composition is storedfor 12 months or greater.
 35. A composition according to claim 31,wherein the composition is stored for 3 months or greater.
 36. Acomposition according claim 31, wherein the composition is stored for 1week or greater.
 37. A composition according to claim 31, wherein thecomposition is stored for 1 day or greater.
 38. A composition accordingclaim 1, wherein the virus is stable to freeze-thawing.
 39. A method ofproducing a composition for the preservation of a virus, said methodcomprising preparing a liquid composition comprising a virus, apolycationic lipid that has a hydrophilic moiety that includes two ormore groups derived from positively charged amino acids, and acryoprotectant, wherein the polycationic lipid has the followingchemical formula:

or a salt thereof, wherein X is a positively-charged hydrophilic moiety;Y is a spacer having a carbon chain length equivalent to 1 to 30carbon-carbon single covalent bonds or is absent; and R₁, R₂, and R₃ arethe same or different acyl groups of a fatty acid.
 40. A methodaccording to claim 39, wherein the lipid has the following chemicalformula:


41. A method according to claim 39, wherein the cryoprotectant is apoly-hydroxy compound.
 42. A method according to claim 41, wherein thepoly-hydroxy compound is a sugar.
 43. A method according to claim 42,wherein the sugar is sucrose, trehalose, dextrose, lactose, maltose,glucose or any combination of these sugars.
 44. A method according toclaim 39, wherein the composition further includes a surfactant.
 45. Amethod according to claim 44, wherein the surfactant is a non-ionicsurfactant.
 46. A method according to claim 45, wherein the non-ionicsurfactant is a molecule that includes an oxyethylene group and ahydroxy group.
 47. A method according to claim 46, wherein the non-ionicsurfactant is polysorbate 80 or polyethylene glycol
 400. 48. A methodaccording to claim 39, wherein the virus is a virus derived from one ormore of the group consisting of Adenoviridae, Atadenoviridae,Herpesviridae, Poxviridae, Parvoviridae, Papoviridae, Orthohepadnavirus,Parvoviridae, Birnaviridae, Reoviridae, Flaviviridae, Picornaviridae,Togaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Arenaviridae,Bunyaviridae, Orthomyxoviridae, and Retroviridae.
 49. A method accordingto claim 48, wherein the virus is derived from the Adenoviridae familyof viruses.
 50. A method according to claim 39, wherein the virus ispurified by a method including chromatography or centrifugation.
 51. Amethod according to claim 39, wherein the composition is formed bycombining a solution including the virus and the cryoprotectant with asolution including the lipid.
 52. A method according to claim 39,wherein the virus is storage stable.
 53. A method according to claim 39,wherein the virus is stable to freeze-thawing.
 54. A composition for thepreservation of a virus, said composition comprising a virus, apolycationic lipid that has a hydrophilic moiety that includes two ormore groups derived from positively charged amino acids, and acryoprotectant, wherein the virus is storage stable when the compositionis stored as a frozen liquid, and wherein the polycationic lipid has thefollowing chemical formula:

or a salt thereof, wherein X is a positively-charged hydrophilic moiety;Y is a spacer having a carbon chain length equivalent to 1 to 30carbon-carbon single covalent bonds or is absent; and R₁, R₂, and R₃ arethe same or different acyl groups of a fatty acid.
 55. A composition forthe preservation of a virus, said composition comprising a virus, apolycationic lipid that has a hydrophilic moiety that includes two ormore groups derived from positively charged amino acids, and acryoprotectant, wherein the virus is stable to freeze-thawing, andwherein the polycationic lipid has the following chemical formula:

or a salt thereof, wherein X is a positively-charged hydrophilic moiety;Y is a spacer having a carbon chain length equivalent to 1 to 30carbon-carbon single covalent bonds or is absent; and R₁, R₂, and R₃ arethe same or different acyl groups of a fatty acid.